: The HIV-1 envelope proteins are recognized as primary immune targets during infection and have been a major focus of AIDS vaccine development to elicit enduring broadly neutralizing antibody responses. These efforts have largely focused on the gp120 and gp41 ectodomains, based on the assumption that the gp41 intracytoplasmic domain (ICD) was not exposed and important for envelope immunogenicity. Recent studies from our lab and others, however, clearly indicate that the ICD is a major determinant of HIV-1 envelope structure, function, and antigenicity and that the ICD can be a potent target for neutralizing antibodies directed to relatively conserved protein segments. We hypothesize that critical segments of the ICD of HIV-1 gp41 are actually located on the exterior of the viral or cellular lipid bilayer and that these segments are important determinants of Env antigenic and immunogenic properties, both by association with other Env domains and as distinct epitopes for neutralizing antibody responses. We hypothesize further that the ICD can be engineered to enhance AIDS vaccine capacity to produce optimal neutralizing antibody responses. We propose in this application to test this hypothesis in 2 specific aims: (i) To determine the topology of ICD sequences in representative clade B and clade C Envs relative to the viral and cellular lipid bilayers, and (ii) To assess the role of the ICD as a determinant of Env antigenicity and immunogenicity with respect to envelope antibody reactivity and neutralization. The structural studies will utilize high resolution membrane protein topology mapping techniques and antibody reactivity assays to define ICD structure in the context of virus-and cell-associated Env and in the context of novel chimeric proteins containing the HIV-1 ICD fused to the ectodomain of influenza virus HA envelope protein. The immunologic studies will employ experimental immunization of rabbits using our established DNA vaccine procedures with codon optimized HIV-1 full length of truncated Envs or engineered HA-gp41 constructs to isolate ICD immunogenicity. It is anticipated that the results of these studies will for the first time define the topology of the HIV-1 ICD and characterize its potential as an immunogen to elicit broadly neutralizing antibodies. The long range goal of this project is to engineer the ICD to optimize the capacity of Env immunogens to elicit enduring broadly reactive neutralizing antibodies. Lay summary: This proposal evaluates the structure of a relatively uncharacterized segment of the HIV-1 envelope and examines the role of these envelope sequences as determinants of viral neutralization properties and for its potential as a vaccine to enhance the production of neutralizing antibodies. [unreadable] [unreadable] [unreadable]